Comparison method for measuring the hemoglobin content of blood



Aug. 22, 1950 9 M. G. BROWN COMPARISON METHOD FOR MEASURING THE HEMOGLOBIN CONTENT OF BLOOD Filed Nov. 10,1945

INVENTOR.

. MORDEN 6 BROWN A T TORNEY Patented Aug. 22, 1950 COMPARISON METHOD FOR MEASURING THE HEMOGLOBIN CONTENT OF BLOOD Morden G. Brown, Williamsville, N. ,Y., assignor to American Optical Company. Scuthbridge, Mass, a voluntary association of Massachusetts Application November 10, 1945, Serial No. 627,873

6 Claims.

This invention relates to a method for conveniently, precisely and rapidly determining the hemoglobin concentration of blood.

Various conditions of a patients blood have a direct and material influence upon the health of the patient. Accordingly, accurate measurements of any of these conditions of the blood are highly desirable as, for example, measurements of the hemoglobin concentration in the blood which may be used to accurately indicate tion will become apparent from a consideration a normal condition or an abnormal condition of of the detailed description which follows taken the patients blood such as anemia. in conjunction with the accompanying drawings Since the hemoglobin concentration or red wherein a preferred form of the apparatus and pigment of the blood is directly associated with certain steps in the method are shown and dethe patients condition, measurements of said scribed. It will be understood, however, that the concentration are widely used by physicians for invention is not to be limited to the details of diagnoses and for accurate records of individual the disclosure except as defined by the appendcase histories. Such determination of the coned claims since the disclosure is given for purcentration of hemoglobin in the blood is gener- Doses of illustration only. ally madecolorimetrically and can not be made In e dr W S as accurately with untreated Whole blood as with Fig. l is a p p e w Of a p 0f Plates hemolyzed blood since the variations in the numwhich may be used as a part Of t e app ber. of blood cells, size and shape have a direct of he inv ntion, said pla es be n shown in bearing thereon. A colorimetric determination u spaced relation in order to disclose the details ofthe hemoglobin concentration of the blood, Of Construction e accordingly, may be best had when the red pis- 2 i P p e View Of the p s of ment thereof is evenly distributed throughout Fig. 1 shown in clamped relation in a resilient the blood specimen and not concentrated in cells holder d ready for separated by clear plasma. Fig. 3 is a perspective view of the plates like-. Accordingly, it is an object of this invention wise positioned in the hold r. ut having a p ito provide an improved method for subjecting men-receiving area of the lower plate Ofiset Or a specimen of blood, which is in its most conmoved outwardly to a position for receiving a I venient form, oxyhemoglobin, produced merely specimen thereon, and with a treated stick or. by exposure to air, to a treatment to hemolyze rod in position for stirring the specimen on the the said specimen to bring about a uniform disspecimen-receiving plate; tribution of red pigment or hemoglobin therein. Fig. 4 shows an enlarged view of the treated. It is a further object of the invention to form stick of the invention; i this specimen into a layer of uniform predeter- Fig. 5 shows a carrying case or container for mined thickness whereby the light-absorbing receiving a plurality of said sticks or rods, said characteristics of this specimen may be studied 40 container forming a substantially moisture proof and compared against a comparison member or enclosure therefor; and a series of comparison members having known Fig. 6 is an enlarged fragmentary view showing light-absorbing values, to thereby obtain an actwo similar specimen-receiving plates placed tocurate indication of the hemoglobin condition gether for use in forming a blood chamber of of the patients blood. I double thickness. It is also an object of the invention to pro- Referring to the drawings in detail, and parvide convenient means in the form of a blood ticularly Fig. 1, the numeral [0 indicates gen.- chamber assembly comprising a pair of specially erally a lower specimen-receiving plate which formed transparent plates, providing surfaces may be used in combination with a cover plate for receiving and holding a specimen while bel2 of equal size, general shape and thickness. ing hemolyzed, and a clamp for holding these Both of said plates are transparent. The upper members together to produce a relatively thin and lower surfaces I 4 and it of the upper plate layer of predetermined thickness between said are accurately finished to provide optically flat plates, whereby the light-absorbing properties surfaces or parallel planes thereon. The lower maybe easily observed or measured. plate I!) is likewise finished upon its lower sur-.

It is also an object of the invention to pro- 4 ness, whereby a specimen of double predetermined thickness may be examined, such being extremely important for accurate results in cases of extreme anemia.

Further objects and advantages of the invenform spaces for receiving excess blood from theui oi area 24 or 25 when the platesand ll are assembled one upon the other. The-areas 24- and 25 for receiving the blood specimen 30,"as indicated in Fig. 3, are accurately formediso'i as to be disposed a predetermined-distance 'below' l the plane of the side rail portions an'd =22, and are arranged parallel to said plane; thereby. forming a space of uniform predetermined thickness between each of these areas and the cover plate l2 when the cover plate is positioned there- 20 over-:1

The plates l0 and l2 maybe conveniently-and firmly held in an assembled positionbya clamporzholder 32 whichis formed-of resilient mate'rial; such asbronzeystainless steelor the'like -gg, and has a base portion 34-=to' which a handle 3510f suitable size' and shape may log-riveted or otherwise secured. The holder 'is ipI'0Vided with a pair of spaced resilient side-members 38 extendingin"substantially parallel relation from thezbase portion 34, and-one of said sidemem-'- ber's is'provided with an upperrpair of 'arms' 40 i and the other of said side'members is provided with a lower pair'of arms 40 arranged in laterally-spaced relation to provide upper and lower unobstructed areas or. spaces 42' aligned with: specimen-receiving areas 24' 'and- 25 -throughi" which the specimen 30may be"observed: The distance between the members38 -'is somewhat1 greater than the combined thicln'iess 'ofthemlates hand |2,fas is clearly indicated' in'Fig. 2, so thatlth'e lower pair of armslfl may be1provided-" with inwardly curved end portions 44', asshown, to engage the outer surface of one'iof the'plates' and press both plates into fir m' engagement .With 5 the other pair of resilient arms 40." Thus;- when these. plates are-in an assembled positionin the if holder, they will be firmly 'clampe'd fitogethe'r' and iprovide between the area M ou 25 and. the

cover'plate a space of predetermined thickness;-

A 'pa'ir of indentations 45 are pressed 'into 'th'e base portion 34': near opposite ends thereofiso that they will be engaged bythe inner. edges- 0f thenplates l0 and I 2' when-in theholderfi32fandl-ii space these plates from theinner wall or surface-- 5 of the base portion 34 thus.preVentingany bIOQd which may be on these edges from" touchingaand adhering to said inner surface.

Fig. 3 shows the-specimen plate I0 and -cover plate l2' in an assembled position clamped be cineular.

A treated rod or stick 46 is shown in con- 9 ceiving an heinolytic agent 52. By the words' inert material as used above and hereinafter in the specification and claims is meant a material which will not be materially affected by the hemolytic agent forming the coating 52 and which will not alter the condition of the blood specimen under examination-.

The rod or-stick may be made'of any preferred cross-sectional shape desired, but as shown is While the proportions or dimensions of- -this rod or stick may vary somewhat, a preferred size would be between 1% and 1% inches in length and from to 5 of an inch in thickness. The-tapered'end portions 58 of the rod orstick fifiis'foi'med so as to receive the coating 52*upo'nthe outer end thereof and support said: coating in'. a space entirely within the planes containing the parallel sides of the stick, whereby th'e hemolytic material will be normally spaced from or above any supporting surface upon which the'stick rests,-or-spacecl from the side \vallsof a container or the like used therewitl1,-or spaced: from other similar fsticks when bunched -to-' getherin said container.

A suitable hemolytic agent which has been found=to give satisfactory results for forming'thev layer or-coating 52 may comprise saponin' which may be applied to-the'stick by forming a tacky solution of saponindn water or other suitable-- solvent and dipping the ends of the rods 01'" sticks" it-into said solution. The=coating on the" rod or stick is then air-dried or dried in an oven" at temperaturesnot exceeding 200 F. Improved results may be obtained by adding -a small amount-of sodium oxalate or-other anti-'coagu-= lant to thehe'molytic agenttobe applied to therods" or sticks, which "additional material will' materially lessen-the tendency of the blood specimento coagulate-.

Itnis de'sirous-that-the treated rod or stick 46 w be formed of stiff, durable and inert material in' order' -that the material-forming :the hemolytic agent will not be affected therebyi A convenient" amount: of hemolytic agent uponthe tapered end' of'the-stick has been found torbe-provid'edi by 'dippingeach stick to a depth of from; to JA ofi an: :inch' into said hemolytic solution *wi-th 101' :1 without the anti-coagulant 1 as desired, A con-n tainer 54 is shown in Fig.5,said-'container beinm; formedof a substantially moisture proof-plastic material toserve as =a suitable means for storing: and transporting-la plurality of sticks aspart' of the equipment oftheinventions Relatively long; tapered portionsare 'formed"at55 and 58;onthe f twol parts'of:thisseparable container in order? tor-provide a substantially: air tight joint betweens: the two parts; irr ordei'v that moisture or damp-P ness of. the outside I air may be excluded. from the L containerland thus not affect-the condition of their; hmolytic a material: auponi the? ends of .i thea sticks contained'therein. 1

A "preferred method of use of the apparatus'described abdve fonthe determination-of the :hemo-i' globin' content 'ors 'concentration of the: bl'ood specimen comprises puncturing the -finger: or lobe: of :the ear of- 1 the -patient to ob'tain a small quantityof: blood: to be tested, said blood 'being' placed-upon the area--24; or 25, "of -the'= plate! 0-, if when ina separated condition as-shown by Fig. 1 i

or when in' an assembled' position with the plate I D offset or-projecting outwardly of the plate- I 2 1 and-gripped by the 1 resilient arms of theholder as-sh'ow'n by '-Fig2 3,=and thi'shn'ay bedone by'-- or ear lobe. Thespecimen-30 30obtained on'the' H 5 specimen-receiving area is then agitated or stirred by the treated end of the stick 46. When the. specimen is subjected to the hemolyzing agent',isuch as saponin, for a suflicient time dur-' ing the stirring operation, such as for a period ranging from five to fifteen seconds,'a hemolyzing action upon the blood specimen will be produced, which action brings about a rupturing of the membranes surrounding the individual red cells or corpuscles of the blood, allowing the red pigment or hemoglobin thereof to be evenly distributed throughout the specimen and thereby producing a solution of substantially uniform hemoglobin concentration.

-The plate containing the hemolyzed solution so produced may then be covered by the cover plate l2 and pressed or slipped into place in the holder 32, as shown by Fig. 2, or may, if in the oifset position shown in Fig. 3, be pressed inwardly to a position adjacent the cover plate [I so..that, in either case, the prepared solution will be contained between the lower surface It of the plate I2 and the area 24, or 25, of the plate ID as a uniform layer of predetermined thickness. The solution so prepared will tend to spread over the entire specimen-receiving area by capillary attraction between these adjacent slightly spaced surfaces. Any excess solution at the outer edge 0f the plates IE! and I2 may be wiped therefrom and if there is an excess amount on the area when the cover plate is placed over the area 24, or 25, it may flow into the grooves 26 or 28 or to the outer edges of the plates 10 and 42 as the layer of predetermined thickness is formed.

When a layer of uniform thickness of prepared solution has been produced between the plates l0 and I2, these plates may be positioned in the field of a colorimetric device or the like and the light-absorption properties thereof observed and compared with a standard comparison member or a series of standard comparison areas of known light-absorbing values. Thus when a comparison area of known value and of lightabsorbing properties which are the same as the specimerf has been ascertained an accurate indication of the hemoglobin concentration of the specimen may be determined therefrom.

In cases of extreme anemia where the amount of red pigment or hemoglobin in the blood is relatively small the physician may employ to good advantage two specimen-receiving plates ID with the specimen-receiving areas thereon facing each other as shOWn by Fig. 6, thereby forming blood chambers between each adjacent pair of areas of double predetermined thickness as indicated by the letters t and t and, accordingly, the specimen placed therebetween will have double lightabsorbing values which may be, in said cases of extreme anemia, more accurately observed and measured or compared in a colorimetric device, or the like, than could the specimen if it were only half as thick. Obviously, in such a combination of similar plates a blood chamber of single thickness t may readily be formed when desired by merely placing the upper plate up-side-down upon the lower plate since surfaces 24 and 25 are parallel to and equally spaced from the surface I8.

Thus, it will be seen that a convenient, accurate and efficient method and apparatus have been provided for ascertaining the hemoglobin content or concentration of a patients blood.

Having described the invention, I claim:

1. The method of measuring the hemoglobin concentration of a patients blood comprising the steps of applying a specimen of said blood, ex-.

tracted from said patient, upon a surface of a.

transparent specimen-receiving plate, stirring said specimen with an hemolytic agent in air for a-time sufiicient to bring about a rupturing of the membranes of the red cells of the blood and produce a substantially uniform distribution of the pigment thereof throughout said specimen to produce an oxyhemoglobin solution, positioning a transparent cover plate over said specimenreceiving plate and prepared solution so as to produce a layer of uniform predetermined capillary thickness and uniform pigment distribution therebetween, transmitting light through said plates and layer, and matching substantially the light-absorbing properties thereof with a comparison area of predetermined light-absorbing value whose hemoglobin concentration equivalent value vis known for determining therefrom the equivalent hemoglobin concentration of the specimen under observation.

2. The method of measuring the hemoglobin concentration of a patients blood comprising the steps of applying a specimen of said blood, ex-

tracted from said patient, upon a surface of a transparent specimen-receiving plate, stirring said specimen with an hemolytic agent comprising saponin in air for a time sufficient to bring about a rupturing of the membranes of the red cells of the blood and produce a uniform distribution of the pigment thereof throughout said specimen to produce an oxyhemoglobin solution, positioning a transparent cover plate over said specimen-receiving plate and prepared solution so as to produce a layer of uniform predetermined capillary thickness and uniform pigment distribution therebetween, transmitting light through said plates and layer, and matching substantially the light-absorbing properties thereof with a comparison area of predetermined light-absorbing value whose hemoglobin concentration equivalent value is known for determining therefrom the equivalent hemoglobin concentration of the specimen under observation.

3. The method of measuring the hemoglobin concentration of a patients blood comprising the steps of applying a specimen of said blood, extracted from said patient, upon a surface of a transparent specimen-receiving plate, stirring said specimen with hemolytic and anti-coagulating agents in air for a time sufficient to bring about a rupturing of the membranes of the red cells of the blood and to produce a substantially uniform distribution of the pigment thereof throughout said specimen to produce an oxyhemoglobin solution, positioning a transparent cover plate over said specimen-receiving plate and prepared solution so as to produce a layer of uniform solution of predetermined capillary thickness between said plates, transmitting light through said plates and layer, and matching substantially the light-absorbing properties thereof with a comparison area of substantially equal light-absorbing value and whose hemoglobin concentration equivalent value is known.

4. The method of measuring the hemoglobin concentration of a patients blood comprising the steps of applying a specimen of said blood, extracted from said patient, upon a surface of a transparent specimen-receiving plate, stirring said specimen with hemolytic and anti-coagulating agents'comprising saponin in air for a time sufficient to bring about a rupturing of the membranes of the red cells of the blood and to produce a uniform distribution of the pigment thereof F f i thtoughont saiia' specimeni'to proauee an: oxy' hemoglobinsolation, f position-in a ir'aiisparent covet-plate over said" speoimemreceiving plate and prepared solution-so as tdprdduoe a layer of-un-ifoi'm predetermined 'c'apill'a'r'y thickness -be-" tween said plates, transmitting light"thl"0ligh said platesand-la'yei, and -matchi'ng substantially thelight abs'orbing properties-thereof with a com--- parison "area of substantiallyequal light absorb ing "va1ueand' Whore hemoglobin" eoneentration equivalent value is known.

Themethod of measuring the hemoglobin concentration of a patients blood'eomprising' the traeted from said patient, upon 'a surface of a transparent specimen-receivingplate," stirringsaid Spe'cinie'n with hemolytic i and antiwoagulating'u agents comprising saponin' and sodinm oxalate in air'for'a time suifieient'to' bring about -20"; of the blood and to produ'ce'an' oxyliemoglobina rupturing of the membranesofthe Ted "cells uniform distribution" of" the pigment thereof throughout'said Specimen" 'to' producea solution; positioning a transparer'it cov'e'rzplate' over said" specimen-receiving plate and prepared solution" so as to produce a layefof uniform predetermined" capillary thickness betw'een'said plates; trans known.

6. The -methodof -measuring the hemoglobin concentration of a patien-ts blood comprising the steps of placing a --pai1-'ofspecimen-receiving plates together in face-to face relation with the specimen-receiving areas of each plate' upon ad jacent sides of said plates and with the specimenreceiving area of one plate projecting outwardly-7' 8e beyommiuikemrea or theiotherzpiate surficiemw ly to expdse Balffiyap'plyifig a specimen '01 b10603 ext'i aefleii from' sai'd patient} -u on said exposed area}, apprymg anmemolyticagent to sam -speck men}; stirring said speoimen and' agent =in' air su'ffioi'n'tly-' to'- p1 bd\c a substa-ntially lnnifoim disti'ibii tiori ofi pigment throughout said specl men .ee ais oxyhemogiebm somtim; movin rsei'eii projecting plafie mtov anwoperative position so as td-{eause theispecimen-re'ceiving areas 'of' said'i platesi40 lie ad jacem; eaeh 3 other and form a. P 1ay'ei of f sdlution of uniform predetermii'i'd' capillai y tlii'eknessz aand matchingnsubstamianyx' the light-absorbing properti'es' ofi said layei' with .a :bdifipansenrareaiiof substantially equal? lightabsorbing slalhe and whose hem'oglobin' 'eoncen-z trationequival'ent'value isknown;

M'ORDENTGJBROWIU 1 REFERENGES CITED The-following Tefeienees" are of" record'inth fil ofthipziitehti UNITED STATES" PATENTS Quinn-REFERENCES 'i eciifi cai Manual; by iwari De artment, 'on f Methods for" Labo'ra'tony Teeh'niCiansfTM8-227; published in 19 1-1; byqoi ernnient Printin'g Offioe'f page;99, paragraph 97. 

